Transcript Profiles of Leaf Development Indicate Co-ordinated Cell
Development and Maturation
To obtain a broader understanding of developmental processes in wild
type plants we conducted a transcriptomic comparison of the base, middle
and tip regions of seven day old leaves. One-way analysis of variance
identified 440 transcripts that were significantly differentially
abundant (P< 0.05) in the different leaf regions. Transcripts
were subjected to hierarchical clustering analysis which revealed five
major clusters (Fig. 3). Cluster A comprised 69 transcripts that
exhibited a gradient of abundance from low in the leaf base to high in
the tip. This cluster included six transcripts encoding transcription
factors homologous to Arabidopsis transcripts that have been shown to
have roles in leaf development (Table S1). MLOC_74058.1 exhibits
homology to an Arabidopsis transcription factor NGATHA3 (AT1G01030)
involved in the control of leaf shape and expressed in leaf tips under
the control of TCP (TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING
CELL FACTOR) transcription factors (Ballester et al., 2015). The latter
family were represented by MLOC_14785.1 which exhibited homology to
Arabidopsis TCP5 (AT5G60970). A gene (MLOC_70809.1) encoding a
homologue of Arabidopsis GATA, NITRATE-INDUCIBLE, CARBON METABOLISM
INVOLVED (GNC) transcription factor (AT5G56860) that regulates stomatal
development, greening and chloroplast development (Bastakis, Hedtke,
Klermund, Grimm, & Schwechheimer, 2018; Klermund et al., 2016; Zubo et
al., 2018) was also present in cluster A.
Two of the transcription factors identified within cluster A (Fig. 3)
were associated with the control of senescence in response to metabolic
signals. AK373121 exhibits homology to an Arabidopsis zinc finger family
protein METHYLENE BLUE SENSITIVITY 1 (MBS1; AT3G02790) responsible for
acclimation or cell death in dose-dependent response to1O2 (Shumbe et al., 2017) while
MLOC_64240.2 and MLOC_53744.1 both share homology to AT1G56010
encoding NAC1, a senescence associated transcription factor under the
control of auxin (Kim et al., 2011). Further evidence for the
upregulation of senescence-associated processes in the leaf tip was the
increased abundance of transcripts (AK370424, MLOC_47161.1) encoding
proteins with homology to AUXIN-INDUCED IN ROOT CULTURES 3 (AT2G04160)
and SENESCENCE-ASSOCIATED GENE 12 (AT5G45890; SAG12), endopeptidases
required for protein turnover (James et al., 2018; Neuteboom,
Veth-Tello, Cludesdale, Hooykaas, & van der Zaal, 1999). Furthermore,
several transcripts (MLOC_56129.2, MLOC_57630.1, AK374126) encoding
proteins homologous to proteins required for ubiquitin mediated protein
turnover exhibited greatest abundance in the leaf tip (Table S1).
Cluster B was the largest of the clusters comprising 187 transcripts
that exhibited a gradient of abundance from high to low from the leaf
base to the leaf tip (Fig. 3). Seventeen transcripts encoding
transcription factors were identified, several of which exhibited
homology to Arabidopsis transcripts with functions in photomorphogenesis
and development. Two transcripts (AK364144, MLOC_73144.4) showed
homology to Arabidopsis auxin response factors (AT4G30080, AT1G19220;
ARF) with functions in leaf morphogenesis and development (Liu, Jia,
Wang, & He, 2011; Schuetz, Fidanza, & Mattsson, 2019). Similarly,
AK376150 and AK365841 are homologues of Arabidopsis genes encoding
INDETRMINATE DOMAIN 15 (AT2G01940) and GATA TRANSCRIPTION FACTOR 2
(AT2G45050), with functions in leaf morphogenesis and
photomorphogenesis, respectively (Cui et al., 2013; Luo et al., 2010).
As described below, a feature of cluster B were large numbers of
transcripts associated with lipid and wax metabolism. Interestingly, we
identified a transcript (AK364135) with homology to an Arabidopsis
transcript encoding the class I TCP transcription factor TCP14
(AT3G47620). In Arabidopsis class I TCP transcription factors including
TCP14 are master regulators of cuticle biosynthesis (Camoirano et al.,
2020) and are required for the induction of genes involved in
gibberellin biosynthesis and cell expansion in response to temperature
(Ferrero, Viola, Ariel, & Gonzalez, 2019). Similarly, several
transcripts in cluster B were associated with polyphenol metabolism and
a transcription factor (AK361986) homologous to Arabidopsis MYB4
(AT4G38620) which functions in the control of flavonoid biosynthesis
(Wang et al., 2020) was also identified in this cluster.
Consistent with the hypothesis that cells at the leaf base were
undergoing division and expansion, 14 transcripts categorised as cell
wall associated were identified in cluster B (Table S1). These included
several transcripts (AK248822.1, AK356936, MLOC_36439.1, MLOC_43237.1,
MLOC_12096.1, MLOC_73204.3) with homology to transcripts encoding
Arabidopsis expansins with a well-established role in cell wall
loosening, leaf initiation and subsequent growth (Marowa, Ding, & Kong,
2016). A further three transcripts (MLOC_61972.1, AK361522, AK361278)
encoded xyloglucan endotransglycosylases (XTHs) that function in cell
expansion by loosening cell walls (Rose, Braam, Fry, & Nishitani,
2002). Furthermore, transcripts encoding two pectin modifying enzymes, a
methylesterase (MLOC_54267.1) and an acetylesterase (MLOC_55102.5)
were highly abundant in the leaf base.
Transcripts associated with lipid metabolism were also highly
represented within cluster B, consistent with the hypothesis that active
cuticle biosynthesis is occurring in the basal portion of the leaf. For
example, MLOC_67622.1 and MLOC_45058.1 both exhibited homology to
Arabidopsis transcripts encoding 3-KETOACYL-COA SYNTHASE 6 (KCS6,
AT1G68530). Plants carrying mutations in KCS6 exhibited
significant reductions in branched and unbranched long chain alkanes and
alcohols in cuticular wax (Buster, & Jetter, 2017). Similarly,
AK252678.1 shared homology with AT5G43760 encoding KCS20 a very long
chain fatty acid synthase required for cuticular wax and root suberin
biosynthesis (Lee et al., 2009). Two transcripts (MLOC_54056.1 and
AK370579) shared homology with AT1G02205 encoding ECERIFERUM 1, mutants
of which exhibit similar defects to KCS6 mutant lines (Buster, &
Jetter, 2017). Taken together these data suggest that the basal portion
of the leaf comprises actively dividing/expanding cells.
Cluster C (Fig. 3) comprised 43 transcripts that were abundant at the
leaf base, scarce in the middle section of the leaf with intermediate
abundance in the leaf tip. Cluster D comprised 127 transcripts that
exhibited a similar high to low abundance profile during leaf maturation
as observed for cluster B. However, the expression gradient in cluster D
was considerably greater than observed for cluster B. Like cluster B,
cluster D contained several transcripts encoding proteins associated
with cell wall metabolism including expansins, XTHs and pectin modifying
enzymes (Table S1). Cluster D additionally contained transcripts
encoding proteins required for cellulose biosynthesis where two
transcripts (MLOC_66568.3, MLOC_68431.4) exhibited homology to
Arabidopsis cellulose synthases (AT5G44030, AT5G17420) and a further two
transcripts (MLOC_7722.1, AK370617) exhibited homology to an
Arabidopsis transcript encoding the membrane anchored COBRA-LIKE 4
(AT5G15630) which plays a key function in cellulose deposition (Brown,
Zeef, Ellis, Goodacre, & Turner, 2005).
Furthermore, cluster D contained transcripts associated cell expansion,
cell polarity, organ patterning and development. MLOC_53132.1 exhibited
significant homology to Arabidopsis transcripts (AT4G08950) encoding
EXORDIUM, a brassinosteroid responsive gene that acts upstream of
wall-associated kinases and expansins to promote cell expansion
(Schröder, Lisso, Lange, & Müssig, 2009). Indeed, a transcript
(AK364262) with homology to the Arabidopsis transcript encoding
WALL-ASSOCIATED KINASE 2 (AT1G21270) required for turgor driven cell
expansion was also identified (Kohorn et al., 2006). Several transcripts
associated with vascular development and patterning were present.
MLOC_58644.1 and AK359559 exhibited homology to Arabidopsis AT2G34710
and AT5G62880 encoding PHABULOSA and ROP11, respectively which play
roles in xylem patterning during early cellular differentiation (Müller
et al., 2016; Nagashima et al., 2018). Furthermore, MLOC_69397.2
homologous to an Arabidopsis transcript (AT1G79430) encoding ALTERED
PHLOEM DEVLOPMENT with a role promoting phloem development was present
(Bonke, Thitamadee, Mähönen, hauser, & Helariutta, 2003). Finally, a
transcript encoding a basic-helix-loop-helix transcription factor
(MLOC_55768.1) with similarity to an Arabidopsis transcript encoding
bHLH93 (AT5G65640) was also present. This transcription factor interacts
with FAMA which controls differentiation of guard cells in the leaf
epidermis (Ohashi-Ito and Bergmann, 2006). Taken together these results
imply active cellular expansion and differentiation in the base of the
leaf and indicate that these processes are complete in the more mature
leaf regions. Transcripts in cluster E displayed a similar pattern of
abundance to those in cluster C with minimum abundance in the mid region
of the leaf.